At least 15 enterococci species exist, but only two of them are commonly associated with clinical infection, namely, Enterococcus faecalis, which is responsible for 80% infections caused by enterococci, and Enterococcus faecium. The Gram-positive bacterium Enterococcus faecalis is a natural inhabitant of the mammalian gastrointestinal tract, and is commonly found in soil, sewage, water, and food, frequently through faecal contamination (Klare, I., Werner, G. and Witte, W. Contrib. Microbiol. 2001, 8, 108-22).
Enterococci are important nosocomial pathogens causing a variety of infections including nosocomial bacteremia, endocarditis, as well as urinary tract, surgical wound, and foreign body infections.
Sensitive strains of these bacteria can be treated with ampicillin and vancomycin. Nevertheless, some enterococci are intrinsically resistant to β-lactam-based antibiotics (some penicillins and virtually all cephalosporins) as well as many aminoglycosides. In the last two decades, particularly virulent strains of Enterococcus that are resistant to vancomycin (Vancomycin-resistant Enterococcus, or VRE) have emerged in nosocomial infections of hospitalized patients especially in the US. The increasing occurrence of enterococcal strains resistant to multiple antibiotics underscores the necessity to improve the understanding of the pathogenesis of infection (Murray, B. E. N. Engl. J. Med. 2000, 342, 710-721; Theilacker, C., Krueger, W. A., Kropec, A. and Huebner, J. Vaccine 2004, 22 Suppl 1, S31-8).
Teichoic acids can be found in the cell wall of gram-positive bacteria, such as Staphylococci, Streptococci, Bacillus, Clostridium, Corynebacterium and Listeria, and appear to extend to the surface of the peptidoglycan layer. Teichoic acids are not found in the gram-negative bacteria.
They can be covalently linked to N-acetylmuramic acid of the peptidoglycan layer, to the lipids of the cytoplasmic membrane, or to a terminal D-alanine in the tetrapeptide crosslinks between N-acetylmuramic acid units.
The main function of teichoic acids is to provide rigidity to the cell-wall by attracting cations such as magnesium and sodium. Teichoic acids are usually, but not always, substituted with D-alanine ester residues, giving the molecule zwitterionic properties. These zwitterionic teichoic acids are suspected ligands for toll-like receptors 2 and 4. Teichoic acids also assist in regulation of cell growth by limiting the ability of autolysins to break the β(1-4) bond between the N-acetyl glucosamine and the N-acetylmuramic acid. Teichoic acids serve as an attachment site for some parasites. Destruction of the bacteria and release of the teichoic acid into the bloodstream may cause fever, blood vessel dilation and possibly shock and subsequent death. Teichoic acid can also be used by bacteria to attach to mucosal membranes.
Lipoteichoic acid (LTA) is a major constituent of the cell wall of Gram-positive bacteria. It consists of teichoic acids, long chains of glycerol or ribitol phosphate and is anchored to the lipid bilayer via a glyceride. LTA bound to targets can interact with circulating antibodies and activate the complement cascade to induce a passive immune kill phenomenon.
Teichoic acids and lipoteichoic acids have been considered strong exogenous pyrogens, i.e. they belong to the substances that may lead to a feverish reaction in a human after a bacterial infection by gram-positive bacteria. They are most likely recognized by the toll-like receptor TLR-2 that is expressed on monocytes and dendritic cells, B- and T-lymphocytes and macrophages. Furthermore, they lead to the excretion of cytokines, and therefore are one factor for the inflammatory reaction following such an infection.
Due to their antigenic properties, they have also been proposed as interesting candidates for the development of synthetic vaccines.
U.S. Pat. No. 7,011,826 describes a vaccine for the prevention of lactic acidosis in a vertebrate, said vaccine comprising at least one isolated microorganism, or fragment or fragments thereof, wherein said microorganism is capable of producing lactic acid within the gut of said vertebrate, and wherein said microorganism is selected from the group consisting of: Clostridium-like species, Prevotella-like species, Bacteroides-like species, Enterococcus-like species, Selenomonas species.
In order to provide more efficient strategies to effectively treat and/or prevent infection in vertebrates caused, at least in part, by enterococci, new antigenic bacterial targets are needed which could both be used in new and improved vaccination strategies, as well as in the development and production of respective vaccines.
As a part of the search for carbohydrate virulence factors and the development of alternative treatments such as glycoconjugate vaccines to combat enterococcal infections, the present invention fulfils these need by providing new capsular polysaccharides, wall lipoteichoic acids and lipoteichoic acids isolated from the cell wall of enterococci.
Thus, the objects of the present invention in a first aspect thereof are solved by an enterococcal cell wall component consisting of
and modified derivatives thereof, and pharmaceutically acceptable salts thereof.
This enterococcal cell wall component (in the following also designated as “enterococcal antigen”) provides a new antigenic target for the development of more efficient strategies to effectively treat and/or prevent infection in vertebrates caused, at least in part, by enterococci, allow for improved vaccination strategies, and allow the development and production of respective vaccines, such as glycoconjugate vaccines.
According to the present invention, the term a “modified derivative” or “modified derivatives” shall include chemically or enzymatically modified enterococcal antigens according to the formula I as above, wherein said modified derivative maintains its function as an enterococcal antigenic determinant and/or to the same, or substantially the same, extent as the enterococcal antigen according to formula I. Preferably, said modified derivative exhibits a quantitatively increased immunological reaction, compared to a non-modified enterococcal antigen. Such increase of the immunological reaction can be detected with immunological assays known in the art.
Examples for modified derivatives are preferably compounds of formula I that are modified to include a linker group in order to be coupled or conjugated to other chemical entities. These linker groups are known in the state of the art, and usually are immunologically inactive, i.e. do not interfere with the immunological properties of the enterococcal antigen. Other modifications include the addition of chemical moieties of the enterococcal antigen in order to carry a detectable label, such as chelating groups or enzymatic groups. Furthermore, peptide (e.g. His) or other “labels” or “tags” can be added in order to be able to purify and/or use the enterococcal antigen in diagnostic assays.
Finally, the enterococcal antigen can include chemical modifications, for example at the rings of the sugar components of the enterococcal antigen, wherein the antigen can be modified to replace an existing side group with either H, an unsubstituted, monosubstituted or polysubstituted C1-C18-alkyl, wherein said alkyl can be straight, branched or cyclic, alkenyl, an unsubstituted, monosubstituted or polysubstituted aryl or heteroaryl residue, an unsubstituted, monosubstituted or polysubstituted benzene group, an acyl group, such as formyl, acetyl, trichloroacetyl, fumaryl, maleyl, succinyl, benzoyl, or a branched or heteroatom or aryl substituted acyl group, an alkoxy substituent, such as —OMe, —OEt, —OnPr, -iPr, —OnBu, —OiBu, —OsecBu, —OtBu, whose alkyl group can be branched, straight or cyclic, an alkyl group bound via a sulphur atom such as —SMe, —SEt, or a sulfonyl group, such as —SO3H, —SO2Me, —SO2CF3, —SO2C6H4CH3 or SO2C6H4CH2Br, or a nitrogen substituent, such as NH2, NHR, —NRR′ (with R, R′=alkyl, alkenyl or aryl as above), NC or —NO2, or fluoro, chloro, bromo, iodine, —CN or a heterosubstituent. As mentioned above, these derivatives are preferably included in order to improve the solubility of the antigen, increase the immunological effect of said antigen (preferably quantitatively), and/or to allow the compound to be coupled to other moieties, e.g. in order to be coupled to a surface (such as a well or chip), and/or to be used in diagnostic assays.
Another aspect of the invention relates to a method for producing the enterococcal cell wall component according to the present invention, wherein said method comprises isolating said enterococcal cell wall component from a bacterial fraction, or comprising synthesizing said antigen, at least in part, through chemical synthesis. Isolation can include purifying said cell wall component from bacterial fractions to be substantially free of other bacterial components, but can also include the isolation as part of certain bacterial fractions, such as cell wall fractions including other parts of the cellular wall, as described herein.
Another aspect of the invention relates to an antibody, preferably a monoclonal antibody or antigenic fragment thereof, that specifically recognizes an enterococcal antigen according to the present invention. The term “antibody” shall include both monoclonal or polyclonal antibodies, recombinant antibodies or fragments thereof, such as Fab and the like, as well as human or humanized antibodies.
Another aspect of the invention then relates to a method for producing the antibody according to the present invention, comprising immunizing a mammal, preferably a rabbit, with an enterococcal cell wall component according to the present invention, or a with the pharmaceutical composition according to the present invention, and preferably the vaccine according to the present invention. Respective methods are known to the person of skill, and are disclosed in the state of the art.
Yet another aspect of the present invention then relates to a method for producing the antibody according to the present invention, comprising generating hybridoma cells producing said antibody as a monoclonal antibody, or comprising a recombinant production of said antibody in a host cell. Respective methods are known to the person of skill, and are disclosed in the state of the art
Still another important aspect of the present invention then relates to the use of the enterococcal antigen according to the present invention as an antigen in the production of antibodies that are specific for said antigen.
Another aspect of the invention then relates to a pharmaceutical composition, comprising at least one enterococcal antigen according to the present invention and/or at least one antibody according to the present invention, together with a pharmaceutically acceptable carrier and/or excipient.
Particularly preferred is a pharmaceutical composition according to the present invention, wherein said composition comprises a cell wall component according to formula I, namely a WTA.
Further preferred is a pharmaceutical composition according to the present invention, wherein said composition is formulated as a vaccine, in particular against infections caused by enterococci, in particular antibiotic resistant enterococci, such as VRE strains, preferably of E. faecalis. Most preferred is a pharmaceutical composition according to the present invention, wherein said cell wall component according to formula I is present in a glycoconjugate vaccine.
The WTA according to the present invention (either present as the antigen alone or in an extract or bacterium as described herein) is preferably used for an enterococci-vaccine, either for active or passive immunization.
Thus, according to the invention, there is provided a pharmaceutical composition, and in particular a vaccine, for the prevention of enterococcal infections in a vertebrate, said pharmaceutical composition comprising at least one new enterococcal antigen according to the present invention, optionally together with a pharmaceutically acceptable carrier, adjuvants and/or diluent.
Typically, the vaccine can comprise live or dead intact cells of at least one enterococcal strain, preferably of E. faecalis, comprising the enterococcal antigen of the invention. More typically, the vaccine comprises cell lysate from at least one of said enterococcal strains as comprising the enterococcal antigen or antigens. Even more typically, the vaccine comprises a crude enterococcal antigen mixture or purified a enterococcal antigen or enterococcal antigens from at least one of said enterococcal strains, preferably E. faecalis. Still more typically, the vaccine comprises a fraction of the cell wall and associated proteins as enterococcal antigen of at least one of said enterococcal strains. The vaccine may also be comprised of a combination of one of the components. Most preferred is a glycoconjugate vaccine comprising an enterococcal antigen according to the present invention. Another aspect relates to a pharmaceutical composition or vaccine, wherein the enterococcal antigen as included has been produced, at least in part, through chemical synthesis. The methods for purifying the selected bacterial fractions containing enterococcal antigens are known to the person of skill, and further described herein.
Typically, the vertebrate is a monogastric, herbivore or ruminant animal or human subject. Even more typically, the vertebrate is selected from the group consisting of human, non-human primate, murine, bovine, ovine, equine, porcine, caprine, leporine, avian, feline and canine. More typically, the vertebrate is selected from the group consisting of human, ovine, camelids, porcine, bovine, equine or canine.
The pharmaceutical composition can be formulated for administration via intramuscular, subcutaneous, topical or other parenteral route. In general, the microorganisms of the present invention are commensal in nature. Thus, oral administration is generally not an effective route of vaccination, and as a consequence, administration via an intramuscular, subcutaneous topical or other parenteral route is preferred. The vaccine may also include cytokines, such as: G-CSF, GM-CSF, interleukins or tumour necrosis factor alpha, used singly or in combination.
The pharmaceutical composition may also include an adjuvant. More typically, the adjuvant is selected from the group consisting of Freunds Complete/Incomplete Adjuvant, Montenide Macrol Adjuvant, Phosphate Buffered Saline and Mannan oil emulsions, saponins (QuiLA) dextran (dextran sulphate, DEAE-Dextran), aluminum compounds (Imject Alum), N-acetylglucosamiyl-N-acetylmuramyl-L-alanyl-D-isoglutamine (Gerbu adjuvant). More typically, the adjuvant is selected from the group as described in the Vaccine 1995, vol 13, p 1203; 1993 vol 11 p 293; and 1992 vol 10 p 427, the disclosures of which are incorporated herein by reference.
Yet another important aspect of the present invention then relates to an enterococcal cell wall component (enterococcal antigen) according to the present invention, the antibody according to the present invention, or the pharmaceutical composition according to the present invention for use in the treatment of diseases, such as bacterial infections, in particular enterococcal infection, such as nosocomial bacteremia infection, endocarditis, urinary tract infections, surgical wound infections, and foreign body infections.
Yet another important aspect of the present invention then relates to the use of the enterococcal cell wall component according to the present invention, the antibody according to the present invention, or the pharmaceutical composition according to the present invention (i.e. the enterococcal antigens) for the treatment against bacterial infections or for the preparation of a medicament against bacterial infections, in particular enterococcal infection, such as nosocomial bacteraemia infection, endocarditis, urinary tract infections, surgical wound infections, and foreign body infections, in particular caused by antibiotic resistant enterococci, such as VRE strains, such as E. faecalis. 
According to yet another preferred embodiment of the invention, there is provided a method for inducing an immune response against at least one enterococcal strain comprising the enterococcal antigen of the present invention in a vertebrate, said method comprising administering to said vertebrate an immunologically effective amount of the vaccine in accordance with the invention, or a pharmaceutical composition in accordance with the invention.
According to yet another preferred embodiment of the invention, there is provided a method for treating or preventing a bacterial infection in a vertebrate, comprising administering to said vertebrate a therapeutically effective amount of the enterococcal cell wall component according the present invention, the antibody according to the present invention, or the pharmaceutical composition according to the present invention.
Preferred is a method according to the present invention, wherein said bacterial infection is an enterococcal infection, such as nosocomial bacteraemia infection, endocarditis, a urinary tract infection, surgical wound infection, and foreign body infection, and is particular caused by antibiotic resistant enterococci, such as a VRE strain, and in particular E. faecalis. 